Metabolic syndrome is a clinical condition characterized by a cluster of pathologies that includes obesity and impaired regulation of carbohydrate and fat metabolism. Lifestyle modifications producing weight loss improves biomarkers of metabolic syndrome, but the high rate of recidivism with these strategies has prompted evaluation of alternative nutritional approaches. Dietary restriction of the essential amino acid methionine by 80% (MR) in rodents limits the accumulation of body fat despite an increase in food intake. A significant increase in energy expenditure compensates for increased food intake and accounts for the reduced accumulation of body fat. A number of studies conducted by our lab and others show that dietary MR also improves carbohydrate and fat metabolism. Preliminary data underscored in the current application suggest that MR may activate general control non-derepressible 2 (GCN2) kinase, a protein which is activated when any one of the 9-10 essential amino acids (EAAs) in the diet are removed. However, there are a number of fundamental differences between studies which completely remove EAAs (100%) and our studies with 80% MR suggesting that GCN2 may not explain all of the responses. Our hypothesis is that GCN2 activation reduces the expression of genes for fat synthesis in the liver and increases the expression of genes related to fat synthesis and oxidation in adipose tissue, and that these changes are responsible for at least part of the increase in energy expenditure following MR. The objective of the proposed studies will be to determine 1) which components of the overall responses to MR require GCN2, 2) if changes in carbohydrate and fat metabolism in liver and adipose tissue are necessary for increasing metabolism and 3) if MR decreases pre- existing obesity, fatty liver and insulin resistance in adult mice. In our first series of studies,we will use mice which have a genetic deletion of GCN2 to determine the role of this protein on the response to MR. We will then treat fat and liver cells isolated from a different group of wild-type and GCN2 null mice with media containing the amino acid levels found in blood after a prior MR study. These studies will allow us to determine if the different responses to MR for fat-producing genes in liver and adipose tissue are direct and if these effects are caused by GCN2. In our second series of studies, we will place mice with an adipose tissue-specific deletion of the gene for stearoyl CoA desaturase 1 (SCD1) on MR to determine if an inhibition of the synthesis of fats reduces metabolism in adipose tissue and decreases overall energy expenditure. In our final study, we will determine if MR reverses pre-existing obesity, fatty liver and insulin resistance in mice after a high fructose diet. These studies will help us to determine how MR is detected and generates its effects and will refine our approach to accomplish my long-term goal of conducting these studies in humans as a therapeutic strategy for obesity and other metabolic disease-related conditions. PUBLIC HEALTH RELEVANCE: The results of the proposed studies will lead to a more thorough understanding of the integrated responses of the liver and adipose tissue to the intake of specific nutrients such as amino acids in the diet. These studies will also provide insight into the capacity of nutrients to functionally remodel the storage and production capacity of fat in adipose tissue. These studies have the potential to enhance our understanding of the causes of obesity and could lead to the development of dietary or pharmacological strategies to reduce the progression of weight-gain and the burden of obesity-related diseases in the United States and throughout the world.